target_offtarget

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##all viruses - target and off target

library(tidyverse)

── Attaching core tidyverse packages ─── tidyverse 2.0.0 ──
✔ dplyr     1.1.4     ✔ readr     2.1.5
✔ forcats   1.0.0     ✔ stringr   1.5.1
✔ ggplot2   3.5.1     ✔ tibble    3.2.1
✔ lubridate 1.9.4     ✔ tidyr     1.3.1
✔ purrr     1.0.2     ── Conflicts ───────────────────── tidyverse_conflicts() ──
✖ dplyr::filter() masks stats::filter()
✖ dplyr::lag()    masks stats::lag()
ℹ Use the ]8;;http://conflicted.r-lib.org/conflicted package]8;; to force all conflicts to become errors

library(ggpubr)
library(tidyplots)

Attaching package: ‘tidyplots’

The following object is masked from ‘package:ggpubr’:

    gene_expression

library(ggthemes)
library(scales)

Attaching package: ‘scales’

The following object is masked from ‘package:purrr’:

    discard

The following object is masked from ‘package:readr’:

    col_factor

Figure 1 - linear model

Read-in data

#Linear model for TE data - separated by virus
#November 2024

#viral read depth
#use bowtie2 data

library(tidyverse)
library(broom)
library(boot)

####read counts/normalised read counts####

#metadata

metadata <-
  read.csv(
    "metadata/sampleIDs_TESpikeIn.csv",
    header = TRUE
  )

metadata2 <- metadata |>
  select(Sample.ID, Number.of.read.pairs..quality.adaptor.trimmed.) |>
  rename(Sample_id = Sample.ID) |>
  rename(QC_reads = Number.of.read.pairs..quality.adaptor.trimmed.)

#import and combine read count files
#deduplicated and non-deduplicated

counts_dedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

counts_nodedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

counts_bt_all <- rbind(counts_dedup_bt, counts_nodedup_bt)

counts_reads <- left_join(counts_bt_all, metadata2, by = "Sample_id")

####read depths####

#import and combine read depth files

depth_dedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readdepth_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

depth_nodedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readdepth_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

Format data

depths_bt_all <- rbind(depth_dedup_bt, depth_nodedup_bt)

depths_reads <- left_join(depths_bt_all, metadata2, by = "Sample_id") |>
  mutate(genome_structure = case_when((
    virus == "Human_adenovirus_40" |
      virus == "Human_betaherpesvirus"
  ) ~ "DNA",
  ,
  .default = "RNA"
  ))

####linear model for spike in viruses - mean read depth####

depths_reads_sub <- depths_reads |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  filter(type == "dedup_TE") |>
  mutate(log_depth = log10(mean_depth)) |>
  mutate(log_viral_load = log10(Viral.load))

#inspect data
hist(depths_reads_sub$mean_depth)
hist(depths_reads_sub$Viral.load)
hist(depths_reads_sub$log_depth)
hist(depths_reads_sub$log_viral_load)

summary(depths_reads_sub$log_depth)
summary(depths_reads_sub$log_viral_load)

model

#all viruses together

lm1 <-
  glm(log_depth ~ log_viral_load * virus,
      data = depths_reads_sub,
      family = "gaussian")

summary(lm1)

glm.diag.plots(lm1)

pred <- predict(lm1, type = "response")

rsq <- function (x, y) {
  cor(x, y) ^ 2
}

rsq(depths_reads_sub$log_depth, pred)

##split by viruses

cols <- c("#4477AA",
          "#66CCEE",
          "#228833",
          "#CCBB44",
          "#EE6677",
          "#AA3377")

facet_names <- c(
  "Human_adenovirus_40" = "Human adenovirus 40",
  "Human_betaherpesvirus" = "Human betaherpesvirus",
  "Human_respiratory_syncytial_virus" = "Human respiratory syncytial virus",
  "Influenza_B_virus" = "Influenza B virus",
  "Mammalian_orthoreovirus3" = "Mammalian orthoreovirus 3",
  "Zika_virus" = "Zika virus"
)

virus <- (unique(depths_reads_sub$virus)) %>%
  rep(., each = 2) %>%
  data.frame()

list_models <- 
  depths_reads_sub |>
  group_split(virus) |>
  map( ~ lm(log_depth ~ log_viral_load, data = .))

lm_tidy <- 
  map(list_models, broom::tidy) %>%
  do.call(rbind.data.frame, .) %>%
  cbind(virus, .) %>%
  rename(virus = ".") %>%
  select(virus, term, estimate) %>%
  pivot_wider(names_from = "term", values_from = "estimate")

lm_summary <- depths_reads_sub |>
  group_by(virus) |>
  summarise(
    Intercept = lm(log_depth ~ log_viral_load)$coefficients[1],
    Coeff_x1 = lm(log_depth ~ log_viral_load)$coefficients[2],
    R2 = summary(lm(log_depth ~ log_viral_load))$r.squared,
    pvalue = summary(lm(log_depth ~ log_viral_load))$coefficients["log_viral_load", 4]
  )

lm_combined <- 
  left_join(lm_tidy, lm_summary, by = "virus")

Make plot

Final plot

#ggsave("figures/compare_spike_ins_atcc/model_readdepth_dedup.png")

ggsave("figures/manuscript_figure_2025/PDF/Figure_1.pdf",
       width = 8,
       height = 5)

ggsave("figures/manuscript_figure_2025/PNG/Figure_1.png",
       width = 8,
       height = 5)

——————–

Figure S1

Read counts

### Read-in data

#ATCC genomes updated version
#October 2024

#viral load vs read count normalised using different methods, comparing deduplicated and non-deduplicated
#use bowtie2 data

####read counts/normalised read counts####

#metadata

metadata <- read.csv("metadata/sampleIDs_TESpikeIn.csv", header = TRUE)

metadata2 <- metadata |>
  select(Sample.ID, Number.of.read.pairs..quality.adaptor.trimmed.) |>
  rename(Sample_id = Sample.ID) |>
  rename(QC_reads = Number.of.read.pairs..quality.adaptor.trimmed.)

#import and combine read count files
#deduplicated and non-deduplicated

counts_dedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

counts_nodedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

counts_bt_all <- rbind(counts_dedup_bt, counts_nodedup_bt)

counts_reads <- left_join(counts_bt_all, metadata2, by = "Sample_id")

#normalise by both raw read count and genome length - should be the same as mean read depth

counts_reads_norm <- counts_reads |>
  mutate(norm_counts1 = matched / QC_reads) |>
  mutate(norm_counts2 = matched / QC_reads / length) |>
  mutate(norm_counts3 = matched / length) |>
  mutate(genome_structure = case_when((
    virus == "Human_adenovirus_40" |
      virus == "Human_betaherpesvirus"
  ) ~ "DNA",
  .default = "RNA"
  ))

Read depths

####read depths####

#import and combine read depth files

depth_dedup_bt <-
  read_tsv(
    "data_TE/TE_sequencing_experiment_readdepth_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv"
  )

Rows: 95 Columns: 12── Column specification ───────────────────────────────────
Delimiter: "\t"
chr (5): virus, Sample_id, Background, mapper, type
dbl (7): Viral load, mean_depth, median_depth, min, max...
ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

depth_nodedup_bt <-
  read_tsv(
    "data_TE/TE_sequencing_experiment_readdepth_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv"
  )

Rows: 95 Columns: 12── Column specification ───────────────────────────────────
Delimiter: "\t"
chr (5): virus, Sample_id, Background, mapper, type
dbl (7): Viral load, mean_depth, median_depth, min, max...
ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

depths_bt_all <- rbind(depth_dedup_bt, depth_nodedup_bt)

depths_reads <- 
  depths_bt_all |>
  left_join(metadata2, by = "Sample_id") |>
  mutate(genome_structure = case_when((
    virus == "Human_adenovirus_40" |
      virus == "Human_betaherpesvirus"
  ) ~ "DNA",
  .default = "RNA"
  )) |>
  rename(Viral.load = `Viral load`)

Format data

#change labels in facet plots

facet_names <- c("dedup_TE" = "Deduplicated",
                 "nodedup_TE" = "Non-Deduplicated")

cols <- c("#4477AA",
          "#66CCEE",
          "#228833",
          "#CCBB44",
          "#EE6677",
          "#AA3377")

Make plots

#plot viral read counts (non normalised)

read_count <- 
  counts_reads_norm |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = Viral.load,
    y = log10(matched),
    colour = virus
  )) +
  geom_point() +
  geom_smooth(aes(group = virus, linetype = genome_structure), se = FALSE) +
  facet_grid( ~ type, labeller = as_labeller(facet_names)) +
  theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 0, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_color_manual(
    values = cols,
    labels = c(
      "Human adenovirus 40",
      "Human betaherpesvirus",
      "Human respiratory syncytial virus",
      "Influenza B virus",
      "Mammalian orthoreovirus 3",
      "Zika virus"
    )
  ) +
  scale_linetype_manual(values=c("dotted", "solid")) + 
  ylab("Log (Viral Reads)") +
  xlab("Genome copies") + 
  scale_x_log10(labels = scales::trans_format("log10", scales::label_math()))

read_count_norm1 <- 
  counts_reads_norm |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = Viral.load,
    y = log10(norm_counts1),
    colour = virus
  )) +
  geom_point() +
  geom_smooth(aes(group = virus, linetype = genome_structure), se = FALSE) +
  facet_grid( ~ type, labeller = as_labeller(facet_names)) +
  theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 0, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_color_manual(
    values = cols,
    labels = c(
      "Human adenovirus 40",
      "Human betaherpesvirus",
      "Human respiratory syncytial virus",
      "Influenza B virus",
      "Mammalian orthoreovirus 3",
      "Zika virus"
    )
  ) +
  scale_linetype_manual(values=c("dotted", "solid")) + 
  ylab("Log (viral reads/cleaned reads)") +
  xlab("Genome copies") + 
  scale_x_log10(labels = scales::trans_format("log10", scales::label_math()))

#normalise by raw read count and genome length

read_count_norm2 <- 
  counts_reads_norm |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = (Viral.load),
    y = log10(norm_counts2),
    colour = virus
  )) +
  geom_point() +
  geom_smooth(aes(group = virus, linetype = genome_structure), se = FALSE) +
  facet_grid( ~ type, labeller = as_labeller(facet_names)) +
  theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 0, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_color_manual(
    values = cols,
    labels = c(
      "Human adenovirus 40",
      "Human betaherpesvirus",
      "Human respiratory syncytial virus",
      "Influenza B virus",
      "Mammalian orthoreovirus 3",
      "Zika virus"
    )
  ) +
  scale_linetype_manual(values=c("dotted", "solid")) + 
  ylab("Log (viral reads/cleaned reads/genome length)") +
  xlab("Genome copies") + 
  scale_x_log10(labels = scales::trans_format("log10", scales::label_math()))


read_depths <- 
  depths_reads |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = Viral.load,
    y = log10(mean_depth),
    colour = virus
  )) +
  geom_point() +
  geom_smooth(aes(group = virus, linetype = genome_structure), se = FALSE) +
  facet_grid( ~ type, labeller = as_labeller(facet_names)) +
  theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 0, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_color_manual(
    values = cols,
    labels = c(
      "Human adenovirus 40",
      "Human betaherpesvirus",
      "Human respiratory syncytial virus",
      "Influenza B virus",
      "Mammalian orthoreovirus 3",
      "Zika virus"
    )
  ) +
<<<<<<< HEAD

Error: unexpected input in:
"  ) +
<<"

Final plot

——————–

Figure S2

Read-in data / Make plots

#metadata

metadata <-
  read.csv(
    "metadata/sampleIDs_TESpikeIn.csv",
    header = TRUE
  )

metadata2 <- metadata |>
  select(Sample.ID, Number.of.read.pairs..quality.adaptor.trimmed.) |>
  rename(Sample_id = Sample.ID) |>
  rename(QC_reads = Number.of.read.pairs..quality.adaptor.trimmed.)

#import and combine read count files
#deduplicated and non-deduplicated

counts_dedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

counts_nodedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

counts_bt_all <- rbind(counts_dedup_bt, counts_nodedup_bt)

counts_reads <- left_join(counts_bt_all, metadata2, by = "Sample_id")

#normalise by both raw read count and genome length - should be the same as mean read depth

counts_reads_norm <- counts_reads |>
  mutate(norm_counts1 = matched / QC_reads) |>
  mutate(norm_counts2 = matched / QC_reads / length) |>
  mutate(norm_counts3 = matched / length) |>
  mutate(genome_structure = case_when((
    virus == "Human_adenovirus_40" |
      virus == "Human_betaherpesvirus"
  ) ~ "DNA",
  .default = "RNA"
  ))

####compare library capture pool####

cols3 <- c("#228833", "#AA3377")

facet_names <- c("dedup_TE" = "Deduplicated",
                 "nodedup_TE" = "Non-Deduplicated")

metadata3 <- 
  metadata |>
  select(Sample.ID, Pool.for.sequencing) |>
  rename(Sample_id = Sample.ID) |>
  rename(Pool = Pool.for.sequencing)

counts_pool <- left_join(counts_reads_norm, metadata3, by = "Sample_id")

Make plots

pool_counts_sum <- 
  counts_pool |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = as.character(Viral.load),
    y = log10(matched),
    colour = Pool
  )) +
  geom_boxplot() +
  facet_grid( ~ type, labeller = as_labeller(facet_names)) +
  theme_few() +
  theme(
    # axis.title.x = element_blank(),
    # axis.text.x = element_blank(),
    legend.title = element_blank(),
    axis.title.y = element_text(size = 10),
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  ylab("Log (Viral Reads)") + xlab("Genome copies") +
  scale_color_manual(values = cols3, labels = c("Pool 1", "Pool 2")) + 
  scale_x_discrete(labels = c(bquote(10^{2}), bquote(10^{3}), bquote(10^{5})))
  

# pool_readcounts_norm <- 
#   counts_pool |>
#   filter(Background != "p6") |>
#   filter(Background != "control") |>
#   ggplot(aes(x = virus, y = log10(norm_counts1))) +
#   geom_boxplot() +
#   facet_grid(Pool ~ type) +
#   theme_few() +
#   theme(
#     axis.title.x = element_blank(),
#     # axis.text.x = element_blank(),
#     axis.title.y = element_text(size = 10),
#     panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
#     panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
#   ) +
#   ylab("log10(viral reads/cleaned reads)")

pool_norm1_sum <- 
  counts_pool |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = as.character(Viral.load),
    y = log10(matched),
    colour = Pool
  )) +
  geom_boxplot() +
  facet_grid( ~ type, labeller = as_labeller(facet_names)) +
  theme_few() +
  theme(
    # axis.title.x = element_blank(),
    # axis.text.x = element_blank(),
    legend.title = element_blank(),
    axis.title.y = element_text(size = 10),
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray") 
  ) +
  ylab("Log (viral reads/cleaned reads)") + xlab("Genome copies") +
  scale_color_manual(values = cols3, labels = c("Pool 1", "Pool 2")) + 
    scale_x_discrete(labels = c(bquote(10^{2}), bquote(10^{3}), bquote(10^{5})))

# pool_readcounts_norm2 <- 
#   counts_pool |>
#   filter(Background != "p6") |>
#   filter(Background != "control") |>
#   ggplot(aes(x = virus, y = log10(norm_counts2))) +
#   geom_boxplot() +
#   facet_grid(Pool ~ type) +
#   theme_few() +
#   theme(
#     axis.title.x = element_blank(),
#     # axis.text.x = element_blank(),
#     axis.title.y = element_text(size = 10),
#     panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
#     panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
#   ) +
#   ylab("log10(viral reads/cleaned reads/genome length)")


pool_norm2_sum <- 
  counts_pool |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = as.character(Viral.load),
    y = log10(matched),
    colour = Pool
  )) +
  geom_boxplot() +
  facet_grid( ~ type, labeller = as_labeller(facet_names)) +
  theme_few() +
  theme(
    # axis.title.x = element_blank(),
    # axis.text.x = element_blank(),
    legend.title = element_blank(),
    axis.title.y = element_text(size = 10),
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  ylab("Log (viral reads/cleaned reads/genome length)") + xlab("Genome copies") +
  scale_color_manual(values = cols3, labels = c("Pool 1", "Pool 2")) + 
    scale_x_discrete(labels = c(bquote(10^{2}), bquote(10^{3}), bquote(10^{5})))


#import and combine read depth files

depth_dedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readdepth_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

depth_nodedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readdepth_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

depths_bt_all <- rbind(depth_dedup_bt, depth_nodedup_bt)

depths_reads <- 
  left_join(depths_bt_all, metadata2, by = "Sample_id") |>
  mutate(genome_structure = case_when((
    virus == "Human_adenovirus_40" |
      virus == "Human_betaherpesvirus"
  ) ~ "DNA",
  .default = "RNA"
  ))

depths_pool <- left_join(depths_reads, metadata3, by = "Sample_id")

# pool_readdepths <- 
#   depths_pool |>
#   filter(Background != "p6") |>
#   filter(Background != "control") |>
#   ggplot(aes(x = virus, y = log10(mean_depth))) +
#   geom_boxplot() +
#   facet_grid(Pool ~ type) +
#   theme_few() +
#   theme(
#     axis.title.x = element_blank(),
#     axis.text.x = element_text(angle = 45, hjust = 1),
#     axis.title.y = element_text(size = 10),
#     panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
#     panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
#   ) +
#   ylab("Log (mean read depth)")

pool_depths_sum <-
  depths_pool |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = as.character(Viral.load),
    y = log10(mean_depth),
    colour = Pool
  )) +
  geom_boxplot() +
  facet_grid( ~ type, labeller = as_labeller(facet_names)) +
  theme_few() +
  theme(
    # axis.title.x = element_blank(),
    # axis.text.x = element_blank(),
    legend.title = element_blank(),
    axis.title.y = element_text(size = 10),
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  ylab("Log (mean read depth)") +
  xlab("Genome copies") + 
  scale_x_discrete(labels = c(bquote(10^{2}), bquote(10^{3}), bquote(10^{5}))) +
  scale_color_manual(values = cols3, labels = c("Pool 1", "Pool 2"))

Final plot

ggarrange(
  pool_counts_sum,
  pool_norm1_sum,
  pool_norm2_sum,
  pool_depths_sum,
  nrow = 2,
  ncol = 2,
  common.legend = TRUE, 
  align = "hv"
)


#ggsave("figures/compare_spike_ins_atcc/pool_compare_viruses_sum.png",width=10,height=7)

ggsave("figures/manuscript_figure_2025/PDF/Figure_S2.pdf",
       width = 12,
       height = 8)

ggsave("figures/manuscript_figure_2025/PNG/Figure_S2.png",
       width = 12,
       height = 8)

——————–

Figure S3

##plots of read counts and viral read counts
#November 2024

#metadata

metadata <-
  read.csv(
    "metadata/sampleIDs_TESpikeIn.csv",
    header = TRUE
  )

metadata2 <- metadata |>
  select(Sample.ID, Number.of.read.pairs..quality.adaptor.trimmed.) |>
  rename(Sample_id = Sample.ID) |>
  rename(QC_reads = Number.of.read.pairs..quality.adaptor.trimmed.)

#import read count files

counts_dedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

counts_nodedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

#import viral reads mapped (to calculate proportions)

viral_reads_dedup <-
  read.csv(
    "data_TE/total_virus_mapped_reads_per_sample_dedup_atcc_ref_20241108.csv",
    header = TRUE
  )

viral_reads_nodedup <-
  read.csv(
    "data_TE/total_virus_mapped_reads_per_sample_nodedup_atcc_ref_20241108.csv",
    header = TRUE
  )

cols2 <- c("#BB5566", "#004488")

facet_names <- c("dedup_TE" = "Deduplicated",
                 "nodedup_TE" = "Non-Deduplicated")

reads_metadata_dedup <-
  left_join(counts_dedup_bt, metadata2, by = "Sample_id")

reads_viral_dedup <-
  left_join(reads_metadata_dedup, viral_reads_dedup, by = "Sample_id")

reads_metadata_nodedup <-
  left_join(counts_nodedup_bt, metadata2, by = "Sample_id")

reads_viral_nodedup <-
  left_join(reads_metadata_nodedup, viral_reads_nodedup, by = "Sample_id")

reads_viral_all <- rbind(reads_viral_dedup, reads_viral_nodedup)


reads_plot <- 
  reads_viral_all |>
  group_by(Background, Sample_id, Viral.load, type) |>
  summarise(
    total_reads = (QC_reads * 2),
    viral_reads = total_virus_reads,
    ATCC_reads = sum(matched),
    prop_ATCC = ATCC_reads / total_reads,
    prop_viral = total_virus_reads / total_reads,
    diff = viral_reads - ATCC_reads
  ) |>
  unique()

Make plots

Final plot

——————–

No longer required? Figure S4

Make plots

####read counts/depths split by background####

#change labels in facet plots

facet_names_bg <- c(
  "dedup_TE" = "Deduplicated",
  "nodedup_TE" = "Non-Deduplicated",
  "p2" = "M1",
  "p8" = "M2"
)

#break down by Background x virus

background_counts_reads <- 
  counts_reads_norm |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = Viral.load,
    y = log10(matched),
    colour = virus
  )) +
  geom_point() +
  geom_smooth(aes(group = virus, linetype = genome_structure), se = FALSE) +
  facet_grid(Background ~ type, labeller = as_labeller(facet_names_bg)) +
  theme_few() +
  theme(
    axis.title.x = element_blank(),
    axis.title.y = element_text(size = 10),
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_color_manual(
    values = cols,
    labels =
      c(
      "Human adenovirus 40",
      "Human betaherpesvirus",
      "Human respiratory syncytial virus",
      "Influenza B virus",
      "Mammalian orthoreovirus 3",
      "Zika virus"
      )
  ) +
  scale_x_log10(labels = scales::label_log10()) +
  ylab("Log (Viral Reads)")

backgrounds_counts_reads_norm <- 
  counts_reads_norm |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = Viral.load,
    y = log10(norm_counts1),
    colour = virus
  )) +
  geom_point() +
  geom_smooth(aes(group = virus, linetype = genome_structure), se = FALSE) +
  facet_grid(Background ~ type, labeller = as_labeller(facet_names_bg)) +
  theme_bw() +
  theme_few() +
  theme(
    axis.title.x = element_blank(),
    axis.title.y = element_text(size = 10),
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_color_manual(
    values = cols,
    labels = c(
      "Human adenovirus 40",
      "Human betaherpesvirus",
      "Human respiratory syncytial virus",
      "Influenza B virus",
      "Mammalian orthoreovirus 3",
      "Zika virus"
    )
  ) +
  scale_x_log10(label = scales::label_log10()) +
  ylab("Log (viral reads/cleaned reads)")

backgrounds_counts_reads_norm2 <- 
  counts_reads_norm |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = Viral.load,
    y = log10(norm_counts2),
    colour = virus
  )) +
  geom_point() +
  geom_smooth(aes(group = virus, linetype = genome_structure), se = FALSE) +
  facet_grid(Background ~ type, labeller = as_labeller(facet_names_bg)) +
  theme_few() +
  theme(
    axis.title.x = element_blank(),
    axis.title.y = element_text(size = 10),
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_color_manual(
    values = cols,
    labels = c(
      "Human adenovirus 40",
      "Human betaherpesvirus",
      "Human respiratory syncytial virus",
      "Influenza B virus",
      "Mammalian orthoreovirus 3",
      "Zika virus"
    )
  ) +
  scale_x_log10(labels = scales::label_log10()) +
  ylab("Log (viral reads/cleaned reads/genome length)")

background_read_depths <- 
  depths_reads |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  ggplot(aes(
    x = Viral.load,
    y = log10(mean_depth),
    colour = virus
  )) +
  geom_point() +
  geom_smooth(aes(group = virus, linetype = genome_structure), se = FALSE) +
  facet_grid(Background ~ type, labeller = as_labeller(facet_names_bg)) +
  theme_few() +
  theme(
    axis.title.x = element_blank(),
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray"),
    axis.title.y = element_text(size = 10),
    legend.title = element_blank()
  ) +
  scale_color_manual(
    values = cols,
    labels = c(
      "Human adenovirus 40",
      "Human betaherpesvirus",
      "Human respiratory syncytial virus",
      "Influenza B virus",
      "Mammalian orthoreovirus 3",
      "Zika virus"
    )
  ) +
  scale_x_log10(label = scales::label_log10()) +
  ylab("Log (mean read depth)")

Final plot

——————–

——————–

>>> Figure 2: plots of TE experiment data - genome coverage & per site coverage

Read-in data

counts_nodedup_bt <-
  read_tsv(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv"
  )

Rows: 96 Columns: 9── Column specification ───────────────────────────────────
Delimiter: "\t"
chr (5): virus, Sample_id, Background, mapper, type
dbl (4): length, matched, unmatched, Viral load
ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.

Format data

persite_coverage_nodedup <- 
  nodedup_per_site |>
  rename(Viral.load = `Viral load`) |>
  group_by(Sample_id, virus, Viral.load, Background, type) |>
  filter(coverage > 0) |>
  summarise(genome_coverage = n()) |>
  left_join(lengths) |>
  mutate(percent_coverage = genome_coverage / length)

`summarise()` has grouped output by 'Sample_id', 'virus', 'Viral.load', 'Background'. You can override using the `.groups` argument.Joining with `by = join_by(virus)`

Make plots

Final plot

——————–

——————–

Figure S4

persite_both <- rbind(dedup_per_site, nodedup_per_site)

persite_reads <- left_join(persite_both, metadata2, by = "Sample_id")

persite_norm <- persite_reads |>
  mutate(norm_cov = coverage / QC_reads)

coverage_labels3 <-c("100" = "1e+02",
                    "1000" = "1e+03",
                    "100000" = "1e+05",
                    "p2" = "M1",
                    "p8"= "M2")

persite_norm$`Viral load` <- 
  factor(persite_norm$`Viral load`, 
         labels = c("0",
                    "10^{2}", 
                    "10^{3}",
                    "10^{5}"))


persite_norm |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  filter(type == "dedup_TE") |>
  filter(virus == "Human_respiratory_syncytial_virus") |>
  rename(Viral.load = `Viral load`) |>
  ggplot(aes(x = site, y = coverage, fill = Background)) +
  geom_col() +
  facet_wrap(Viral.load ~ Sample_id, label = label_parsed) +
  ylab("Coverage") +
  ggtitle("Human RSV (deduplicated)") +
  guides(fill = guide_legend(title = "Background")) +
  theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 30, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_y_log10() +
  scale_fill_manual(values = cols2, labels = c("M1", "M2"))

Final plot

ggsave("figures/manuscript_figure_2025/PDF/Figure_S4.pdf",
       width=15,
       height=10)

ggsave("figures/manuscript_figure_2025/PNG/Figure_S4.png",
       width=15,
       height=10)

——————–

Figure S5

####Final plot

ggsave("figures/manuscript_figure_2025/PDF/Figure_S5.pdf",
       width=15,
       height=10)

ggsave("figures/manuscript_figure_2025/PNG/Figure_S5.png",
       width=15,
       height=10)

——————–

Figure S6

####Final plot

ggsave("figures/manuscript_figure_2025/PDF/Figure_S6.pdf",
       width=15,
       height=10)

ggsave("figures/manuscript_figure_2025/PNG/Figure_S6.png",
       width=15,
       height=10)

——————–

Figure S7

HBV <- 
persite_norm |>
  filter(Background != "p6") |>
  filter(Background != "control") |>
  filter(type == "dedup_TE") |>
  filter(virus == "Human_betaherpesvirus") |>
  rename(Viral.load = `Viral load`) |>
  ggplot(aes(x = site, y = coverage, fill = Background)) +
  geom_col() +
  facet_wrap(Viral.load ~ Sample_id, label = label_parsed) +
  ylab("Coverage") +
  ggtitle("Human Betaherpesvirus (deduplicated)") +
  guides(fill = guide_legend(title = "Background")) +
 theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 30, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_y_log10() +
  scale_fill_manual(values = cols2, labels = c("M1", "M2"))

####Final plot

ggsave(plot = HBV, file="figures/manuscript_figure_2025/PDF/Figure_S7.pdf",
       width=15,
       height=10,
       dpi = 600)

ggsave(plot = HBV, file="figures/manuscript_figure_2025/PNG/Figure_S7.png",
       width=15,
       height=10)

——————–

Figure S8

#have to do these differently due to segmentation


FLU_ME1 <- persite_norm |>
  filter(virus == "Influenza_B_virus") |>
  filter(type == "dedup_TE") |>
  filter(Background == "p2") |>
  rename(Viral.load = `Viral load`) |>
  ggplot(aes(
    x = site,
    y = coverage,
    fill = as.character(Viral.load)
  )) +
  geom_col() +
  facet_grid(seg ~ Sample_id) +
  ylab("Coverage") +
  ggtitle("Influenza B virus (Background M1 deduplicated)") +
  guides(fill = guide_legend(title = "Viral load")) +
 theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 30, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_y_log10() +
  scale_fill_manual(values = cols4, label = c(expression("10"^"2"), expression("10"^"3"), expression("10"^"5")))

FLU_ME2 <- persite_norm |>
  filter(virus == "Influenza_B_virus") |>
  filter(type == "dedup_TE") |>
  filter(Background == "p2") |>
  rename(Viral.load = `Viral load`) |>
  ggplot(aes(
    x = site,
    y = coverage,
    fill = as.character(Viral.load)
  )) +
  geom_col() +
  facet_grid(seg ~ Sample_id) +
  ylab("Coverage") +
  ggtitle("Influenza B virus (Background M2 deduplicated)") +
  guides(fill = guide_legend(title = "Viral load")) +
 theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 30, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) +
  scale_y_log10() +
  scale_fill_manual(values = cols4, label = c(expression("10"^"2"), expression("10"^"3"), expression("10"^"5")))


ggarrange(FLU_ME1, FLU_ME2, ncol = 2, common.legend = TRUE)

Warning: log-10 transformation introduced infinite values.Warning: Removed 3867 rows containing missing values or values
outside the scale range (`geom_col()`).Warning: log-10 transformation introduced infinite values.Warning: Removed 3867 rows containing missing values or values
outside the scale range (`geom_col()`).Warning: log-10 transformation introduced infinite values.Warning: Removed 3867 rows containing missing values or values
outside the scale range (`geom_col()`).

####Final plot

ggsave("figures/manuscript_figure_2025/PDF/Figure_S8.pdf",
       width=20,
       height=12,
       dpi=600)

ggsave("figures/manuscript_figure_2025/PNG/Figure_S8.png",
       width=20,
       height=12)

——————–

Figure S9

REO_ME1 <- 
  persite_norm |>
  filter(virus == "Mammalian_orthoreovirus3") |>
  filter(type == "dedup_TE") |>
  filter(Background == "p2") |>
  rename(Viral.load = `Viral load`) |>
  ggplot(aes(
    x = site,
    y = coverage,
    fill = as.character(Viral.load)
  )) +
  geom_col() +
  facet_grid(seg ~ Sample_id) +
  ylab("Coverage") +
  ggtitle("Mammalian orthoreovirus 3 (Background M1 deduplicated)") +
  guides(fill = guide_legend(title = "Viral load")) +
  theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 30, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top",
    panel.grid.major = element_line(linewidth = 0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth = 0.1, color = "lightgray")
  ) +
  scale_y_log10() +
  scale_fill_manual(values = cols4,
                    label = c(
                      expression("10" ^ "2"),
                      expression("10" ^ "3"),
                      expression("10" ^ "5")
                    ))

REO_ME2 <- 
  persite_norm |>
  filter(virus == "Mammalian_orthoreovirus3") |>
  filter(type == "dedup_TE") |>
  filter(Background == "p8") |>
  rename(Viral.load = `Viral load`) |>
  ggplot(aes(
    x = site,
    y = coverage,
    fill = as.character(Viral.load)
  )) +
  geom_col() +
  facet_grid(seg ~ Sample_id) +
  ylab("Coverage") +
  ggtitle("Mammalian orthoreovirus 3 (Background M2 deduplicated)") +
  guides(fill = guide_legend(title = "Viral load")) +
  theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.text.x = element_text(angle = 30, hjust = 1),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top",
    panel.grid.major = element_line(linewidth = 0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth = 0.1, color = "lightgray")
  ) + scale_y_log10() +
  scale_fill_manual(values = cols4,
                    label = c(
                      expression("10" ^ "2"),
                      expression("10" ^ "3"),
                      expression("10" ^ "5")
                    ))

ggarrange(REO_ME1, REO_ME2, ncol = 2, common.legend = TRUE)

Warning: log-10 transformation introduced infinite values.Warning: Removed 2181 rows containing missing values or values
outside the scale range (`geom_col()`).Warning: log-10 transformation introduced infinite values.Warning: Removed 2181 rows containing missing values or values
outside the scale range (`geom_col()`).Warning: log-10 transformation introduced infinite values.Warning: Removed 934 rows containing missing values or values
outside the scale range (`geom_col()`).

####Final plot

ggsave("figures/manuscript_figure_2025/PDF/Figure_S9.pdf",
       width=20,
       height=12,
       dpi=600)

ggsave("figures/manuscript_figure_2025/PNG/Figure_S9.png",
       width=20,
       height=12,
       dpi=600)

——————–

>>>> Figure 3: all viruses - target and off target

Read-in data

# read in metadata

metadata <-
  read.csv("metadata/sampleIDs_TESpikeIn.csv", header = TRUE)

metadata2 <-
  metadata |>
  select(
    Sample.ID,
    Background.sample,
    Viral.load,
    Number.of.read.pairs..quality.adaptor.trimmed.
  ) |>
  rename(Sample_id = Sample.ID) |>
  rename(QC_reads = Number.of.read.pairs..quality.adaptor.trimmed.)

#import viral reads mapped (to calculate proportions)

viral_reads_dedup <-
  read.csv(
    "data_TE/total_virus_mapped_reads_per_sample_dedup_atcc_ref_20241108.csv",
    header = TRUE
  )

viral_reads_nodedup <-
  read.csv(
    "data_TE/total_virus_mapped_reads_per_sample_nodedup_atcc_ref_20241108.csv",
    header = TRUE
  )

#import contingency tables

contingency_dedup <-
  read.csv(
    "data_TE/contingency_table_mapped_virus_reads_per_family_genus_sample_dedup_atcc_ref_20241106.csv",
    header = TRUE
  )

contingency_nodedup <-
  read.csv(
    "data_TE/contingency_table_mapped_virus_reads_per_family_genus_sample_nodedup_atcc_ref_20241106.csv",
    header = TRUE
  )

contaminants <-
  c("Betacoronavirus", "Alphainfluenzavirus", "Gammaretrovirus")

Format data

# pivot longer  
dedup_long <-
  contingency_dedup |>
  filter(!genus %in% contaminants) |>
  filter(genus != "NA") |>
  pivot_longer(cols = A:P,
               names_to = "Sample_id",
               values_to = "count") |>
  mutate(
    target = case_when(
      genus == "Cyclovirus" ~ "off_target",
      genus == "Cardiovirus" ~ "off_target",
      genus == "Kobuvirus" ~ "off_target",
      .default = "on_target"
    )
  ) |>
  mutate(genome_structure = case_when((genus == "Mastadenovirus" |
                                         genus == "Cytomegalovirus") ~ "DNA",
                                      .default = "RNA"
  ))

no_dedup_long <- contingency_nodedup |>
  filter(!genus %in% contaminants) |>
  filter(genus != "NA") |>
  pivot_longer(cols = A:P,
               names_to = "Sample_id",
               values_to = "count") |>
  mutate(
    target = case_when(
      genus == "Cyclovirus" ~ "off_target",
      genus == "Cardiovirus" ~ "off_target",
      genus == "Kobuvirus" ~ "off_target",
      .default = "on_target"
    )
  ) |>
  mutate(genome_structure = case_when((genus == "Mastadenovirus" |
                                         genus == "Cytomegalovirus") ~ "DNA",
                                      .default = "RNA"
  ))

metadata_dedup <- left_join(dedup_long, metadata2, by = "Sample_id")

dedup_viral_reads <-
  left_join(metadata_dedup, viral_reads_dedup, by = "Sample_id") |>
  mutate(total_reads = QC_reads * 2) |>
  mutate(prop_total = count / total_reads) |>
  mutate(prop_viral = count / total_virus_reads)

metadata_no_dedup <- left_join(no_dedup_long, metadata2, by = "Sample_id")

nodedup_viral_reads <-
  left_join(metadata_no_dedup, viral_reads_nodedup, by = "Sample_id") |>
  mutate(total_reads = QC_reads * 2) |>
  mutate(prop_total = count / total_reads) |>
  mutate(prop_viral = count / total_virus_reads)

Make plots

Dedup – viral reads, prop all reads, and prop viral reads

Final plot

…………………….

…………………….

—-Genome medicine results—-

Figure S10

Read-in data

####read counts/normalised read counts####

#import and combine read count files

gm_readcount_dedup <-
  read.table(
    "data_genome_medicine/genome_medicine_TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

gm_readcount_nodedup <-
  read.table(
    "data_genome_medicine/genome_medicine_TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

gm_counts_all <- rbind(gm_readcount_dedup, gm_readcount_nodedup)

read counts

read depth

#import and combine read depth files

depth_dedup_gm <-
  read.table(
    "data_genome_medicine/genome_medicine_TE_sequencing_experiment_readdepth_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

depth_nodedup_gm <-
  read.table(
    "data_genome_medicine/genome_medicine_TE_sequencing_experiment_readdepth_per_sample_and_virus_bowtie2_nodedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

depths_gm_all <- rbind(depth_dedup_gm, depth_nodedup_gm)

——————–

Figure S11

Read-in data

####genome coverage####

unzip(zipfile = "data_genome_medicine/genome_medicine_TE_sequencing_experiment_readdepth_per_site_sample_and_virus_bowtie2_dedup_atcc_ref.tsv.zip", exdir = "data_genome_medicine/")

dedup_per_site <-
  read.table(
    "data_genome_medicine/genome_medicine_TE_sequencing_experiment_readdepth_per_site_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

file.remove("data_genome_medicine/genome_medicine_TE_sequencing_experiment_readdepth_per_site_sample_and_virus_bowtie2_dedup_atcc_ref.tsv")

[1] TRUE

#View(dedup_per_site)

counts_dedup_bt <-
  read.table(
    "data_TE/TE_sequencing_experiment_readcount_per_sample_and_virus_bowtie2_dedup_atcc_ref.tsv",
    sep = "\t",
    header = TRUE
  )

#add length column from read depth file to per site file

lengths <- 
  counts_dedup_bt |>
  select(virus,length) |>
  distinct()

#calculate genome coverage

coverage <- 
  dedup_per_site |>
  group_by(sample_id, virus, Viral.load, Replicate) |>
  filter(coverage > 0) |>
  summarise(genome_coverage = n()) |>
  left_join(lengths) |>
  mutate(percent_coverage = genome_coverage / length)

`summarise()` has grouped output by 'sample_id', 'virus', 'Viral.load'. You can override using the `.groups` argument.Joining with `by = join_by(virus)`

coverage |>
  filter(Viral.load != 0) |>
  ggplot(aes(x = virus, y = percent_coverage)) +
  geom_boxplot(outlier.shape = NA) +
  geom_point(position = position_dodge(width = .75)) +
  facet_grid( ~ as.character(Viral.load)) +
  theme_bw() +
  theme(axis.title.y = element_text(size = 10)) +
  ylab("Genome coverage") +
  theme_few() +
  theme(
    axis.title.x = element_blank(),
    axis.text.x = element_text(angle = 45, hjust = 1),
    axis.title.y = element_text(size = 15),
    axis.text = element_text(size = 12),
    strip.text = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "top",
    panel.grid.major = element_line(linewidth = 0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth = 0.1, color = "lightgray")
  ) +
  scale_y_continuous(limits = c(0, 1)) +
    scale_x_discrete(
    labels = c(
      "Human adenovirus 40",
      "Human betaherpesvirus",
      "Human respiratory syncytial virus",
      "Influenza B virus",
      "Mammalian orthoreovirus 3",
      "Zika virus"
    )
  )

#ggsave("/Users/laura/Dropbox/glasgow/github/te_ug_rodents/figures/genome_medicine/all_genome_coverage.png",width=14,height=6)

#ggsave("/Users/laura/Dropbox/glasgow/github/te_ug_rodents/figures/manuscript_figures_pdf/FigureS12.pdf",width=20,height=6)

*** Final plot ***

#PDF
ggsave(
  "figures/manuscript_figure_2025/PDF/Figure_S11.pdf",
  width = 14,
  height = 6
)

#PNG
ggsave(
  "figures/manuscript_figure_2025/PNG/Figure_S11.png",
  width = 14,
  height = 6
)

——————–

Figure S12

*** Final plot ***

ggsave("figures/manuscript_figure_2025/PDF/Figure_S12.pdf",
       width=12,
       height=10)

ggsave("figures/manuscript_figure_2025/PNG/Figure_S12.png",
       width=12,
       height=10)

…………………….

—-Kobuvirus / Cardiovirus results—-

Figure S13

kobu_depth <-
  read.table(
    "data_kobuvirus/kobuvirus_TE_polyomics_readdepth_20241007.tsv",
    sep = "\t",
    header = TRUE
  ) %>%
  select(!expt) %>%
  select(!Number.of.paired.end.reads..QT.)

cardio_depth <-
  read.table(
    "data_cardiovirus/cardiovirus_denovo_bowtie2_read_depth_per_sample_dedup.tsv",
    sep = ",",
    header = TRUE
  )

cardio_depth_poly <- 
  read.table(
    "data_cardiovirus/cardiovirus_denovo_bowtie2_readdepth_per_sample_dedup_polyomics.tsv",
    sep = ",",
    header = TRUE
  ) |>
  select(-contig_name) |>
  filter(Background %in% c("S18", "S19", "S43")) |>
  select(-Background) |>
  separate(Sample_id, into = c(NA, NA, "Background"), sep = "-", remove = FALSE)

depth_both <- rbind(kobu_depth, cardio_depth, cardio_depth_poly) |>
  mutate(Viral.load = ifelse(is.na(Viral.load), 0, Viral.load))

depth_both$type <- depth_both$type |>
  case_match("dedup_TE" ~ "dedup",
             "dedup_polyomics" ~ "dedup",
             .default = depth_both$type)

depth_both$Sample_id <- depth_both$Sample_id |>
  case_match("RNA-Msp-p2" ~ "M1",
             "RNA-Msp-p8" ~ "M2",
             "RNA-Msp-p6" ~ "M3",
             .default = depth_both$Sample_id
             )

depth_both$virus <- depth_both$virus |>
  case_match("cardiovirus" ~ "Cardiovirus",
             "k97_19971" ~ "Kobuvirus",
             .default = depth_both$virus
             )

cols2 <- c("#BB5566", "#004488")

coverage_labels <- c(
  "k97_19971" = "Kobuvirus",
  "cardiovirus" = "Cardiovirus",
  "p2" = "M1",
  "p8" = "M2"
)

*** Final plot ***

depth_both$Viral.load <- factor(depth_both$Viral.load, labels = c("Shotgun",
    "10^{2}", 
    "10^{3}", 
    "10^{5}"
    ))


# depth_both |>
#   filter(Background != "p6") |>
#   filter(type == "dedup") |>
#   filter(mapper == "bowtie2") |>
#   filter(Viral.load != "NA") |>
#   ggplot(aes(
#     x = Viral.load,
#     y = (mean_depth),
#     colour = Background
#   )) +
#   geom_point() +
#  theme_few() +
#   theme(
#     axis.title.x = element_text(size = 12),
#     axis.title.y = element_text(size = 12),
#     legend.title = element_blank(),
#     legend.position = "none", 
#     panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
#     panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
#   ) +  
#   facet_grid(Background ~ virus, labeller = as_labeller(coverage_labels)) +
#   scale_color_manual(values = cols2) +
#   ylab("Mean read depth") +
#     xlab("Spike-in viral load (gc/ml)") +
#    scale_x_log10(labels = scales::trans_format("log10",
#                                         scales::label_math())) + 
#   scale_y_log10(labels = scales::trans_format("log10",
#                                         scales::label_math()))

# replotting S13 in similar way to S14
depth_both %>%
  filter(Background != "p6") |>
  filter(type == "dedup") |>
  filter(mapper == "bowtie2") |>
  # filter(Viral.load != "NA") |>
  ggplot(aes(x = Sample_id, y = mean_depth, color = Background)) +
  geom_point() +
  ylab("Mean read depth") +
  xlab("Sample ID") +
  facet_grid(virus ~ Viral.load,
             scales = "free_x",
             label = label_parsed) +
  scale_color_manual(values = cols2, labels = c("M1", "M2")) +
 theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "right", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  )+
  scale_y_log10(labels = scales::trans_format("log10",
                                        scales::label_math()))


ggsave("figures/manuscript_figure_2025/PDF/Figure_S13_v2.pdf",
       width=8,
       height=4)

ggsave("figures/manuscript_figure_2025/PNG/Figure_S13_v2.png",
       width=8,
       height=4)

——————–

Figure S14

kobu_per_site <-
  read.table(
    "data_kobuvirus/kobuvirus_TE_polyomics_readdepth_persite_20241007.tsv",
    sep = "\t",
    header = TRUE
  ) %>%
  select(virus,
         seg,
         site,
         coverage,
         n_sites,
         Sample_id,
         Background,
         Viral.load,
         mapper,
         type)

kobu_per_site$Viral.load <- 
  kobu_per_site$Viral.load %>%
  replace_na(., 0)

kobu_per_site$Sample_id <- kobu_per_site$Sample_id %>%
  case_match("RNA-Msp-p2" ~ "M1",
             "RNA-Msp-p8" ~ "M2",
             .default = kobu_per_site$Sample_id)

kobu_per_site$Background <- kobu_per_site$Background %>%
  case_match("p2" ~ "M1",
             "p8" ~ "M2",
             .default = kobu_per_site$Background)

cardio_per_site <-
  read.table(
    "data_cardiovirus/cardiovirus_denovo_bowtie2_read_depth_per_site_and_sample_dedup.tsv",
    sep = ",",
    header = TRUE
  )

cardio_polyomics_per_site <-
  read.table(
    "data_cardiovirus/cardiovirus_denovo_bowtie2_readdepth_per_site_sample_dedup_polyomics.tsv",
    sep = ",",
    header = TRUE
  ) %>%
  select(!contig_name)

#View(cardio_polyomics_per_site)

cardio_TE_polyomics <-
  full_join(
    cardio_per_site,
    cardio_polyomics_per_site,
    by = c(
      "virus",
      "seg",
      "site",
      "coverage",
      "n_sites",
      "Sample_id",
      "Background",
      "Viral.load",
      "mapper",
      "type"
    )
  )

cardio_TE_polyomics$Viral.load <- cardio_TE_polyomics$Viral.load %>%
  replace_na(., 0)

drops <-
  c(
    "RNA-Msp-p1",
    "RNA-Msp-p3",
    "RNA-Msp-p4",
    "RNA-Msp-p5",
    "RNA-Msp-p6",
    "RNA-Msp-p7",
    "RNA-Msp-p9"
  )

cardio_TE_polyomics <- cardio_TE_polyomics %>%
  filter(!Sample_id %in% drops)

cardio_TE_polyomics$Sample_id <- cardio_TE_polyomics$Sample_id %>%
  case_match("RNA-Msp-p2" ~ "M1",
             "RNA-Msp-p8" ~ "M2",
             .default = cardio_TE_polyomics$Sample_id)

cardio_TE_polyomics$Background <- cardio_TE_polyomics$Background %>%
  case_match("S18" ~ "M1",
             "S43" ~ "M2",
             "p2" ~ "M1",
             "p8" ~ "M2",
             .default = cardio_TE_polyomics$Background)

persite_both <- rbind(kobu_per_site, cardio_TE_polyomics)

persite_both$type <- persite_both$type %>%
  case_match("dedup_TE" ~ "dedup",
             "dedup_polyomics" ~ "dedup",
             .default = persite_both$type)

#TE data only - compare coverage between backgrounds/viral loads

cols2 <- c("#BB5566", "#004488")

coverage_labels <- c(
  "100" = "10^{2}",
  "1000" = "10^{3}",
  "100000" = "10^{5}",
  "0" = "Shotgun",
  "Kobuvirus" = "Kobuvirus",
  "Cardiovirus" = "Cardiovirus"
)

coverage_labels2 <- c(
  "100" = "10^{2}",
  "1000" = "10^{3}",
  "100000" = "10^{5}",
  "0" = "Shotgun",
  "A" = "A",
  "B" = "B",
  "C" = "C",
  "D" = "D",
  "F" = "F",
  "G" = "G",
  "H" = "H",
  "I" = "I",
  "J" = "J",
  "K" = "K",
  "L" = "L",
  "M" = "M",
  "N" = "N",
  "O" = "O",
  "P" = "P",
  "ME_P1" = "M1",
  "ME_P2" = "M2"
)

kobu_coverage <- kobu_per_site %>%
  filter(mapper == "bowtie2") %>%
  filter(type == "dedup") %>%
  filter(Background != "p6") %>%
  group_by(Sample_id, Viral.load, Background) %>%
  filter(coverage > 0) %>%
  summarise(genome_coverage = n()) %>%
  mutate(percent_coverage = genome_coverage / 8467) %>%
  mutate(virus = "Kobuvirus")

`summarise()` has grouped output by 'Sample_id', 'Viral.load'. You can override using the `.groups` argument.

cardio_coverage <- cardio_TE_polyomics %>%
  filter(Background != "p6") %>%
  group_by(Sample_id, Viral.load, Background) %>%
  filter(coverage > 0) %>%
  summarise(genome_coverage = n()) %>%
  mutate(percent_coverage = genome_coverage / 6945) %>%
  mutate(virus = "Cardiovirus")

`summarise()` has grouped output by 'Sample_id', 'Viral.load'. You can override using the `.groups` argument.

coverage_both <- rbind(kobu_coverage, cardio_coverage)

coverage_both$Viral.load <- factor(coverage_both$Viral.load, labels = c("Shotgun",
    "10^{2}", 
    "10^{3}", 
    "10^{5}"
    ))

*** Final plot ***

coverage_both %>%
  ggplot(aes(x = Sample_id, y = percent_coverage, color = Background)) +
  geom_point() +
  ylab("Genome coverage") +
  xlab("Sample ID") +
  facet_grid(virus ~ (Viral.load),
             scales = "free_x",
             label = label_parsed) +
  scale_color_manual(values = cols2, labels = c("M1", "M2")) +
 theme_few() +
  theme(
    axis.title.x = element_text(size = 12),
    axis.title.y = element_text(size = 12),
    legend.title = element_blank(),
    legend.position = "right", 
    panel.grid.major = element_line(linewidth=0.1, color = "gray60"),
    panel.grid.minor = element_line(linewidth=0.1, color = "lightgray")
  ) + 
  scale_y_continuous(limits = c(0, 1))

#PDF
ggsave("figures/manuscript_figure_2025/PDF/Figure_S14.pdf", 
       width = 8, 
       height = 4)

#PNG
ggsave("figures/manuscript_figure_2025/PNG/Figure_S14.png", 
       width = 8, 
       height = 4)

——————–

Figure S15

*** Final plot ***

#PDF
ggsave("figures/manuscript_figure_2025/PDF/Figure_S15.pdf",
       width=12,
       height=7)

#PNG
ggsave("figures/manuscript_figure_2025/Png/Figure_S15.png",
       width=12,
       height=7)

Figure S16

*** Final plot ***

#PDF
ggsave("figures/manuscript_figure_2025/PDF/Figure_S16.pdf",
       width=12,
       height=7)


#PNG
ggsave("figures/manuscript_figure_2025/PNG/Figure_S16.png",
       width=12,
       height=7)